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Trial registered on ANZCTR


Registration number
Help: Registration number
ACTRN12626000084381
Ethics application status
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Approved
Date submitted
Help: Date submitted
10/12/2025
Date registered
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22/01/2026
Date last updated
Help: Date last updated
22/01/2026
Date data sharing statement initially provided
Help: Data sharing statement date
22/01/2026
Type of registration
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Prospectively registered

Titles & IDs
Public title
Assessment of Pain Biomarkers in Orthopedic Patients Undergoing Total Knee Arthroplasty
Scientific title
Assessment of Pain Biomarkers in Orthopedic Patients Undergoing Total Knee Arthroplasty
Additional trial identifiers [1] 316145 0
Nil known
Universal Trial Number (UTN)
U1111-1332-7067
Trial acronym
Related trial records

Health condition
Health condition(s) studied:
Knee osteoarthritis 340080 0
Pain biomarkers 340081 0
Knee pain 340082 0
Anxiety 340083 0
Depression 340084 0
Pain catastrophizing 340085 0
Condition category
Condition code
Musculoskeletal 336284 336284 0 0
Osteoarthritis
Mental Health 336285 336285 0 0
Anxiety
Mental Health 336286 336286 0 0
Depression
Mental Health 336287 336287 0 0
Other mental health disorders

Intervention/exposure
Study type
Observational
Patient registry
False
Target follow-up duration
Target follow-up type
Description of the intervention or exposure
Brief name:
Peripheral immune pain biomarker assessment in knee osteoarthritis

Description:
This is an observational study in which no therapeutic intervention is delivered. Participants are observed cross-sectionally with respect to pain, function, psychological measures (PROMs), and peripheral immune biomarkers (blood and synovial fluid samples).

The study planned to enrol 3 groups of participants: 1) 20 patients awaiting total knee arthroplasty (TKA) including 20 painful knees with moderate to severe osteoarthritis (OA), 2) 20 controls, the healthy contralateral pain-free knee of these 20 patients awaiting TKA, and 3) 20 different healthy controls for venous blood measurements (described under "comparator"). This will result in 40 participants, and 60 knees in total: 20 painful knees with osteoarthritis that is awaiting TKA, 20 pain-free contralateral control knees of the same patients, 20 pain-free control knees of different patients.

Preoperatively, at the pre-admission clinic 2-4 weeks before surgery, enrolled TKA-awaiting patients (n= 20) will receive routine venous blood sampling for elective surgery preparations at Noarlunga Hospital. At that same blood collection moment, blood will be collected for this study. Perioperatively, during the elective TKA surgery, synovial fluid will be collected from the painful knee receiving TKA surgery. Similarly, the contralateral pain-free knee will be punctuated for synovial fluid when the patient is still under narcosis.

What is involved for participants:
Participants will undergo a single study visit (healthy controls) or two study-related time points embedded within routine clinical care (TKA patients).
Venous blood sampling:
- Patients awaiting total knee arthroplasty (TKA): one venous blood sample (30 mL) collected preoperatively during the routine pre-admission clinic visit (2–4 weeks before surgery).

Synovial fluid sampling (TKA patients only):
- During the elective TKA procedure, synovial fluid (30 mL) is aspirated from the painful knee immediately after surgical exposure.
- While the patient remains under general anaesthesia, synovial fluid (30 mL) is also aspirated from the contralateral pain-free knee.

All sampling procedures are performed by credentialed clinical staff as part of, or in conjunction with, routine care.

What is being observed / measured:
- Peripheral immune responses measured in peripheral blood mononuclear cells and synovial fluid mononuclear cells, including interleukin-1 beta release following ex vivo Toll-like receptor 2 and 4 stimulation (painCELL assay).
- Immune cell phenotyping using hyperspectral imaging of unstimulated blood and synovial fluid samples (painHS assay).
- Clinical pain severity assessed using the Numeric Rating Scale (NRS).
- Psychological status assessed using validated questionnaires:
Hospital Anxiety and Depression Scale (HADS; approximately 5–10 minutes)
Pain Catastrophizing Scale (PCS; approximately 5 minutes)
- Functional outcomes assessed using:
Oxford Knee Score (OKS)
Knee Injury and Osteoarthritis Outcome Score (KOOS)

Mode and setting of data collection:
All assessments and sample collection occur face-to-face at Noarlunga Hospital/ Flinders Medical Centre in a hospital outpatient or operating theatre setting.

Duration of observation:
Each participant is observed cross-sectionally. Data collection occurs at a single time point for healthy controls, and over a short perioperative window for TKA patients (preoperative clinic visit and day of surgery). There is no longitudinal follow-up.

Personalisation or adaptation:
No intervention, dose adjustment, or personalisation applies, as this is an observational study.

Intervention fidelity or adherence:
Not applicable, as no intervention is delivered.
Intervention code [1] 332757 0
Diagnosis / Prognosis
Description of the comparator or control
The study planned to enrol 20 healthy controls for venous blood measurements. These participants will be age and sex matched healthy individuals with no pain complaints in general at that moment, and no function limitation or pain in any joint (no diagnosed OA in both knees).

All healthy blood-control individuals without pain symptoms in both knees (n= 20) will attend Flinders Medical Centre or Noarlunga Hospital, where similarly venous blood samples will be collected for the study.

What is involved for healthy control participants:
- Healthy controls: one venous blood sample (30 mL) collected during a single outpatient hospital visit.
Control group
Active

Outcomes
Primary outcome [1] 343819 0
This will be assessed as a composite outcome: Measurement of IL-1-beta-concentrations in stimulated peripheral blood mononuclear cells (PBMCs) of TKA and control patients and in stimulated peripheral synovial fluid mononuclear cells (SFMCs) of TKA patients only, parameters include: EC50, Hillslope, Emax.
Timepoint [1] 343819 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Primary outcome [2] 343820 0
This will be assessed as a composite outcome: Hyperspectral imaging of blood samples for TKA and control patients and synovial samples for TKA patients only, to capture cell fluorescence and create quantitative features of immune cell populations (phenotyping of unstimulated cells), parameters include: channel intensity ratios, correlation of pixel intensities, coherence of statistical data from segmented immune cells.
Timepoint [2] 343820 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Secondary outcome [1] 455764 0
Knee pain
Timepoint [1] 455764 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Secondary outcome [2] 455765 0
Knee function
Timepoint [2] 455765 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Secondary outcome [3] 455766 0
Knee function
Timepoint [3] 455766 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Secondary outcome [4] 455767 0
Anxiety
Timepoint [4] 455767 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Secondary outcome [5] 455768 0
Depression
Timepoint [5] 455768 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients
Secondary outcome [6] 455769 0
Pain catastrophizing
Timepoint [6] 455769 0
Preoperatively (before total knee arthroplasty) for TKA patients and at single assessment session for control patients

Eligibility
Key inclusion criteria
TKA patients cohort:
1) awaiting a unilateral primary TKA for symptomatic (painful) advanced OA [Kohn 2016],
2) asymptomatic (pain-free) contralateral knee without function limitation or pain (no or mild osteoarthritis),
3) otherwise good general health,
4) aged 18 years or over
5) completion of all preoperative questionnaires
6) providing informed consent.

Control patients cohort:
1) No pain symptoms in general, no pain in knees or any other joints and no pain from any other complaint or condition.
2) No osteoarthritis in any joint as far as participant is aware of (based on diagnosis from a doctor).
3) Good general health
4) aged 18 years or over
5) completion of all preoperative questionnaires
6) providing informed consent.
Minimum age
18 Years
Maximum age
No limit
Sex
Both males and females
Healthy volunteers
No
Key exclusion criteria
TKA patients cohort:
1) TKA indication other than symptomatic OA for painful knee,
2) painful OA in any other joint,
3) other relevant pain complaints elsewhere,
4) presence of any auto-immune diseases, e.g.: rheumatoid arthritis, lupus, inflammatory bowel disease, psoriasis, etc.
5) one or more of the following comorbidities: diabetes, gout or septic or reactive arthritis in any joint, or any clinical significant renal, hepatic, cardiac disease,
6) a medical history including: fracture or surgery to either of the knees, recent (<3 months) major surgery or significant trauma,
7) presence of an inflammatory process, or clinically significant infection in the last 4 weeks,
8) immunization within the last 4 weeks,
9) current use of immunosuppressant medication such as a steroid, hydroxychloroquine, methotrexate or azathioprine,
10) inability to read or comprehend the written information provided,
11) current use of medications known to affect innate immune responsiveness including amitriptyline, chemotherapy or TNF-a inhibitors
12) current pregnancy.

Control patients cohort:
1) presence of any auto-immune diseases, e.g.: rheumatoid arthritis, lupus, inflammatory bowel disease, psoriasis, etc.
2) one or more of the following comorbidities: diabetes, gout or septic or reactive arthritis in any joint, or any clinical significant renal, hepatic, cardiac disease,
3) a medical history including: fracture or surgery to either of the knees, recent (<3 months) major surgery or significant trauma,
4) presence of an inflammatory process, or clinically significant infection in the last 4 weeks,
5) immunization within the last 4 weeks,
6) current use of immunosuppressant medication such as a steroid, hydroxychloroquine, methotrexate or azathioprine,
7) inability to read or comprehend the written information provided,
8) current use of medications known to affect innate immune responsiveness including amitriptyline, chemotherapy or TNF-a inhibitors
9) current pregnancy.

Study design
Purpose
Screening
Duration
Cross-sectional
Selection
Case control
Timing
Prospective
Statistical methods / analysis
A sample size calculation based on achieving 90% power and a Type 1 error rate of 5% was chosen. The calculation determined that 24 participants in total (12 participants per group) would be required to detect a 2.40 difference in the Least Squares means for EC50 (concentration-response parameter: concentration which produces 50% of the maximal effect, to determine the responsiveness of PBMC and SFMC to LPS and PAM3 stimulation) assuming a within-group standard deviation (SD) of 1.78. The clinically significant difference in EC50 was estimated based on parameters described by Evans et al (2020). To account for a potential 10% loss to follow-up and safety margin with regards to sample analyses, a total sample of 40 participants (20 per group) is to be recruited.

painCELL data analysis
Concentration-response curves from the in vitro innate immune stimulated IL-1ß release from PBMCs and SFMCs, are calculated using GraphPad Prism version 8 (MathWorks; Natick, MA, USA). Differences between pain-free controls (within the same patient for synovial fluid samples and a different patient for blood samples), and painful symptomatic OA patients will be assessed on EC50, Hillslope and “Top” (or Emax). The “Bottom” value will be constrained to a constant of 0. An F-test in GraphPad Prism will be performed to determine if these values should be the same or different between the participant groups. The goodness of fit obtained from the TLR2 stimulation is not sufficient to warrant presentation in the results, owing to lower than expected cytokine expression in the assays.

The painCELL assay cytokine data will be combined with OA severity and NRS-pain reports, as well as other demographics (such as mental state) for each participant in a data file. Additional parameterisation of the data set will be performed to capture the delta change in cytokine release between low and high concentration innate immune challenges, and between these delta values and the steepness of the concentration response.

Analysis will be conducted using IBM SPSS Statistics (Version 27) (or R or Python code). First, a feature selection (dimensionality reduction) step will be performed to remove highly correlated variables, by examining the correlation matrix and applying the 'Bivariate Correlations' function to identify and exclude variables with Pearson Correlation coefficients greater than 0.8. For each specific hypothesis test, data subsets will be created using the ‘Select Cases’ function, to exclude irrelevant subject data (e.g., when testing the model for Healthy synovial sample Controls and the contralateral Painful OA knee, Healthy blood sample Controls data will be excluded).
For analysis, a randomized approach with at least a 20% holdout validation and test dataset will be employed. The dataset will be split using the 'Split File' function, ensuring that the test dataset is not included in the model training phase. For the categorical selection hypotheses, several classification techniques will be applied in parallel, expected to include: Linear Discriminant Analysis (LDA) using the 'Discriminant Analysis' function, Classification and Regression Trees (CART) using the 'Decision Trees' function, K-Nearest Neighbors (KNN) using the 'K Nearest Neighbors' function, Support Vector Machine (SVM) using the 'SVM' extension available through SPSS Modeler, and Random Forest models using the 'Random Trees' extension also available through SPSS Modeler. The best-performing model will be identified based on the highest accuracy and lowest error rate. A confusion matrix will then be created for the best model by applying it to the separate validation dataset using the 'Crosstabs' function. Full outputs of the best-fit model, including classification tables and accuracy measures, will be provided in the Appendix.

painHS data analysis
Hyperspectral images are pre-processed as previously described. This will be followed by cell segmentation, carried out in an automatic approach using a parametric watershed algorithm with a partial set principal components (PCA) image. The channels employed in the PCA segmentation image are found through trial and test. This segmentation protocol will be augmented with additional manual segmentation using the phase contrast image and selecting cells at random. By estimation, this will yield 70-90 cells from each image, belonging to one of four participant groups: 1) blood samples from painful symptomatic OA patients, 2) synovial fluid samples from painful symptomatic OA patients, 3) group 2 contralateral pain-free control knee for synovial fluid samples, 4) blood samples from pain-free controls of different patients.

After each cell is segmented from the background, a number of quantitative features are calculated using the segmented cells to parameterise the cell data, with the primary goal of delineating participant population membership. In particular, for each cell we will calculate ratios of mean channel intensities of pairs, correlation of channel pixel intensities, and coherence of the statistics of the channel pairs. The feature data for all cells will then undergo a singular value decomposition / PCA as described previously, where the new variables satisfied the variance maximisation criteria and are used in creating the unsupervised plots. These uncorrelated variables are then further used for supervised discriminatory analysis. The linear discriminant analysis (LDA) will provide another set of 7 feature variables maximising the separation between the sample populations. These are then optimally projected by using the second linear discriminant analysis onto 2 dimensions to obtain a graphical representation, showing the optimal participant group separation. Subsequently, they are projected again using LDA on a single optimised dimension. Receiver operator curves will be plotted to assess the sensitivity and specificity of the approach.

Recruitment
Recruitment status
Not yet recruiting
Date of first participant enrolment
Anticipated
2/02/2026
Actual
Date of last participant enrolment
Anticipated
2/03/2026
Actual
Date of last data collection
Anticipated
31/08/2026
Actual
Sample size
Target
40
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
SA
Recruitment hospital [1] 28872 0
Flinders Medical Centre - Bedford Park
Recruitment postcode(s) [1] 45131 0
5042 - Bedford Park

Funding & Sponsors
Funding source category [1] 320743 0
Government body
Name [1] 320743 0
National Health and Medical Research Council (NHMRC) - DISCERN Synergy Grant
Country [1] 320743 0
Australia
Funding source category [2] 320745 0
Charities/Societies/Foundations
Name [2] 320745 0
Prins Bernhard Cultuurfonds (Netherlands) – used only for incidental minor expenses if required
Country [2] 320745 0
Netherlands
Primary sponsor type
University
Name
University of Adelaide - Neuroimmunopharmacology Laboratory
Address
Country
Australia
Secondary sponsor category [1] 323397 0
Hospital
Name [1] 323397 0
Flinders Medical Centre
Address [1] 323397 0
Country [1] 323397 0
Australia

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 319279 0
Southern Adelaide Clinical Human Research Ethics Committee
Ethics committee address [1] 319279 0
Ethics committee country [1] 319279 0
Australia
Date submitted for ethics approval [1] 319279 0
12/06/2025
Approval date [1] 319279 0
13/06/2025
Ethics approval number [1] 319279 0
2024/HRE00253

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 146838 0
Prof Ruurd Jaarsma
Address 146838 0
Flinders Medical Centre, department of Trauma and Orthopaedic Surgery, Flinders Dr, Bedford Park, South Australia, 5042
Country 146838 0
Australia
Phone 146838 0
+61882044289
Fax 146838 0
Email 146838 0
[email protected]
Contact person for public queries
Name 146839 0
Margot Aalders
Address 146839 0
Flinders Medical Centre, department of Trauma and Orthopaedic Surgery, Flinders Dr, Bedford Park, South Australia, 5042
Country 146839 0
Australia
Phone 146839 0
+61882044289
Fax 146839 0
Email 146839 0
[email protected]
Contact person for scientific queries
Name 146840 0
Margot Aalders
Address 146840 0
Flinders Medical Centre, department of Trauma and Orthopaedic Surgery, Flinders Dr, Bedford Park, South Australia, 5042
Country 146840 0
Australia
Phone 146840 0
+61882044289
Fax 146840 0
Email 146840 0
[email protected]

Data sharing statement
Will the study consider sharing individual participant data?
Yes
Will there be any conditions when requesting access to individual participant data?
Persons/groups eligible to request access:
• Researchers
Conditions for requesting access:
• Yes, conditions apply:
• Requires a scientifically sound proposal or protocol
• Requires approval by an ethics committee
• Requires a data sharing agreement between data requester and trial custodian or sponsor
What individual participant data might be shared?
• De-identified individual participant data:
• Published results
• Primary outcome(s)
What types of analyses could be done with individual participant data?
• Any type of analysis (i.e. no restrictions on data re-use)
When can requests for individual participant data be made (start and end dates)?
From:
At the end of the study
To:
Not yet decided
Where can requests to access individual participant data be made, or data be obtained directly?
• Email of trial custodian, sponsor or committee: [email protected]
[email protected]

Are there extra considerations when requesting access to individual participant data?
No


What supporting documents are/will be available?

No Supporting Document Provided


Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.